Functional and genomic studies of tidal rhythmicity

Over the past 40 years a considerable amount of knowledge has accumulated on how circadian 24 hour rhythms are generated in a variety of model organisms, from Cyanobacteria to mammals. The circadian clock in higher organisms is composed of a set of positive regulators CLK and BMAL1 and the negative regulators, PER, TIM, CRY2. The positive regulators activate the negative regulators, and the negative regulators feed back in a 24 hour cycle and inhibit the positive regulators. This negative feedback loop generates molecular cycles of 24 hours within a cell. The molecular dissection of the circadian clock is one of the triumphs of modern biology.
However, tidal 12.4 hour behavioural rhythms that reflect the ebb and flow of the tides and are observed in the locomotor behaviour of many intertidal animals have ben completely ignored molecularly. Over the past few years we have been developing a small crustacean, the isopod Eurydice pulchra (marine woodlouse) as a tidal model. These animals show robust tidal cycles of swimming as well as other circadian rhythms. We identified most of the circadian clock genes in Eurydice, and found that by disrupting one of the negative regulators, PER, circadian 24 rhythms were disrupted but the 12.4 hour tidal rhythm of behaviour was completely unaffected. However, when we pharmacologically disrupted the positive regulator CLK, both tidal and circadian cycles were disrupted. Thus the positive regulator appears to control both tidal and circadian rhythms whereas the negative regulator controls only circadian behaviour.
We examined the brain and found four groups of cells that expressed different combinations of the negative regulator PER and the positive regulators CLK and BMA1. One group of cells expressed all three components (the Dorsal cells), whereas another expressed only CLK and BMAL1 (the Laterodorsal cells). If the Dorsal cells determine circadian rhythms and the Laterodorsal cells run the tidal cycle, that would explain our results, in that CLK disruption affects both rhythms but PER disruption affects only circadian oscillations. This model is so simple it's disturbing that nobody has ever thought of it before.
We will develop reagents that recognise all these different proteins - so far we only have good antibodies against PER, CLK and BMAL1 and the latter two come from the fruitfly. The new reagents will allow us to resolve exactly which neurons express which combinations of clock proteins and illuminate our model. We will then disrupt the genes that encode CRY2, TIM, BMAL1 and CLK and examine the circadian and tidal phenotypes, again to see if our model works. Our model requires novel negative tidal regulators, at least one of which must cycle with a 12.4 period to negatively regulate CLK and BMAL1. For the circadian clock we know in Eurydice that TIM provides that 24 h cycling component. How will we find these elusive negative tidal regulators? We cannot find out within a reasonable time without a full annotated genome sequence and indeed the lack of a genome has hindered our efforts considerably. Consequently, we shall use next generation sequencing methods to generate the complete genome of Eurydice, against which we can map and identify all the genes whose mRNAs cycle with a 12.4 hour period. From bioinformatic analyses we shall home in on putative tidal regulators, and generate reagents that will inform us whether their proteins are expressed in the relevant clock neurons. We shall also disrupt the corresponding genes to see whether tidal behaviour is affected. In addition, we will focus in on tidal negative regulators by screening for molecules that sssociate with the Eurydice positive regulators EpCLK-BEpMAL1 In evolutionary terms, it is possible that tidal rhythms evolved before animals became terrestrial so we feel we are on the verge of answering one of the bigger questions in biology.

We have made considerable progress in developing Eurydice pulchra as a model for studying tidal 12.4 hour rhythms. We have developed an automated swimming assay in which we can monitor individual animals which show robust 12.4 h cycles of behaviour. They also show circadian phenotypes in chromatophore pigmentation and in cycling gene expression of Eptimeless. We have identified the canonical clock genes, developed RNAi and used pharmacological methods to disrupt clock gene expression and examine the phenotypic consequences. We have identified clock neurons that express combinations of clock proteins. Using a combination of dsRNAi and pharmacological manipulations, we suggest that a specific dorsal neuron that expresses positive and negative regulators is the putative circadian oscillator, whereas a laterodorsal neurons that expresses only positive regulators is the putative tidal neuron because disrupting the negative regulator affects only circadian rhythms, but disrupting the positive regulator affects both tidal and circadian rhythms. Thus tidal clocks may have a dedicated set of novel negative regulators, at least one of which should have a 12.4 h cycle.
We shall use dsRNAi against relevant clock genes and study rhythmic phenotypes and generate new antisera to further dissect the neurobiology of the two oscillators and further test our model. We shall use modern sequencing methods to produce an annotated Eurydice genome and identify cycling tidal transcripts using RNAseq. Bioinformatic analyses will be used to identify putative tidal regulatory sequences in gene promoters and putative regulatory molecules that we can pursue at the functional level with dsRNAi and spatial/temporal expression patterns in the brain. We shall also use the Y2H system to screen for molecules that physically associate with Eurydice CLK-BMAL1, as these may include the key tidal negative regulators and cross reference them to cycling (and non-cycling) mRNAs.

In the longer term, the main beneficiaries of our work from outside the immediate circle of chronobiologists, will be evolutionary biologists that are generally interested in arthropod evolution, and ecologists who are interested in coastline biology. In addition, as this work may answer one of the bigger questions in biology, should we be successful, our studies might end up in the textbooks with obvious impact for biology education. As the tidal oscillator shares molecular components (through our work) with the circadian oscillator , it also makes tidal organisms relevant to circadian biology which impacts on policymakers concerned about the epidemic of 24 hour lifestyles which have detrimental effects on health.

The industrial production of tidal ragworms for the shrimp food industry is a significant commercial activity in the UK. Millions of these tidal annelids are farmed and what we discover about the tidal oscillator may be of practical benefit. One of the managers of Seabait, one these companies, is Professor Peter Olive (retired, University of Newcastle) who has a special research interest in tidal cycles, and is a former colleague, so our findings will fall on receptive commercial ears.

The public are also very curious about rhythms in general and are responsive to issues concerning body clocks. Within the Genetics Department, a national CETL (Centre of Excellence in Teaching and Learning) for GENIE (Genetics, Education, Networking, Innovation and Excellence), run by Prof Annette Cashmore of our department. A significant activity of GENIE is therefore outreach, and its website attract >22,000 hits per month (http://www.le.ac.uk/genetics/genie/?searchterm=cetl). Within this website is a Virtual Genetics Education Centre for schools and colleges, higher education centres, the general public, as well as health professionals and policymakers. GENIE conducts about 35 meetings/workshops per year, and CPK has contributed to GENIE functions on an ad hoc basis regularly. Consequently, we shall,

1. Work with the GENIE lecturers to add a section on tidal rhythms to the BIORHYTHMS website we have already created as part of other grant related impact activities.
2. Use the opportunities offered to us organised by GENIE, to present our work to various groups, both local and national. We shall be particularly keen to present to health professionals, schools, women's groups etc. However CETL also has regular forums with policymakers (eg House of Lords presentations) and commercial groups (Rotary Clubs) and so we shall deliver lectures to these.